The overall theme of this project is to elucidate the key elements of antigen presentation pathways that influence immunogenicity in vivo and to manipulate these elements to enhance the generation of protective immune responses. There are 3 specific Aims: 1. To study the effects of enhancing ubiquitin and proteasome-dependent processing of antigens expressed from DNA-based vectors on MHC class I presentation and generation of protective immunity in vivo. The hypothesis underlying this Aim is that antigen processing is a key rate limiting step in the presentation of antigens with MHC class I molecules. The goal of this set of experiments is to enhance the processing of antigens in vivo and thereby generate more peptide-MHC complexes and stronger T cell responses. Our experimental approach will be to develop generic modifications of genes encoding antigens that target the modified gene products for processing by the endogenous class I pathway. This approach is based on new insights into the class I processing pathway. These constructs will then be tested for enhancing immunogenicity in vitro, and in vivo, and ultimately for their efficacy in specific disease models. 2. To study the effects of enhancing endosomal and lysosomal processing of antigens expressed from DNA-based vectors on MHC class II presentation and generation of protective immunity in vivo. The hypothesis and goals of this aim are similar to Aim 1 but are directed at enhancing the immunogenicity of antigens for presentation with MHC class II molecules rather than class I molecules. Our experimental approach will be to develop modifications of genes encoding antigens that will target the antigens into appropriate subcellular compartments for them to efficiently enter the MHC class II pathway. This is a particularly important approach for developing effective DNA-based vaccines because most endogenously synthesized antigens to not efficiently gain access to the class II pathway in cells. These constructs will then be tested for enhancing immunogenicity in vitro and in vivo, and for their efficacy in specific disease models. 3. To study the effect of the concomitant administration of cytokine and costimulatory molecules with antigen on T cell priming with recombinant DNA-based vectors. The rationale for this Aim is that the key adhesion molecules and costimulatory factors that are necessary for optical antigen presentation and T cell responses in vitro are known. The expression of many of these factors is regulated by cytokines and cytokines also stimulate antigen presentation pathways. The hypothesis underlying this Aim is that these same factors will be important to the generation of effector T cell responses and memory in vivo and that immunogenicity can be enhanced by upregulating adhesion molecules and costimulatory factors as well as the generation of peptide-MHC complexes t the site of antigen presentation. The experimental approach will be to develop DNA-based vectors that locally coexpress cytokine and/or costimulatory genes together with the antigens developed in Aims and 2. These constructs will then be tested for enhancing immunogenicity and efficacy in vivo.